Genes for asparagine metabolism in Lotus japonicus: differential expression and interconnection with photorespiration.

BACKGROUND: Asparagine is a very important nitrogen transport and storage compound in plants due to its high nitrogen/carbon ratio and stability. Asparagine intracellular concentration depends on a balance between asparagine biosynthesis and degradation. The main enzymes involved in asparagine metabolism are asparagine synthetase (ASN), asparaginase (NSE) and serine-glyoxylate aminotransferase (SGAT). The study of the genes encoding for these enzymes in the model legume Lotus japonicus is of particular interest since it has been proposed that asparagine is the principal molecule used to transport reduced nitrogen within the plant in most temperate legumes. RESULTS: A differential expression of genes encoding for several enzymes involved in asparagine metabolism was detected in L. japonicus. ASN is encoded by three genes, LjASN1 was the most highly expressed in mature leaves while LjASN2 expression was negligible and LjASN3 showed a low expression in this organ, suggesting that LjASN1 is the main gene responsible for asparagine synthesis in mature leaves. In young leaves, LjASN3 was the only ASN gene expressed although at low levels, while all the three genes encoding for NSE were highly expressed, especially LjNSE1. In nodules, LjASN2 and LjNSE2 were the most highly expressed genes, suggesting an important role for these genes in this organ. Several lines of evidence support the connection between asparagine metabolic genes and photorespiration in L. japonicus: a) a mutant plant deficient in LjNSE1 showed a dramatic decrease in the expression of the two genes encoding for SGAT; b) expression of the genes involved in asparagine metabolism is altered in a photorespiratory mutant lacking plastidic glutamine synthetase; c) a clustering analysis indicated a similar pattern of expression among several genes involved in photorespiratory and asparagine metabolism, indicating a clear link between LjASN1 and LjSGAT genes and photorespiration. CONCLUSIONS: The results obtained in this paper indicate the existence of a differential expression of asparagine metabolic genes in L. japonicus and point out the crucial relevance of particular genes in different organs. Moreover, the data presented establish clear links between asparagine and photorespiratory metabolic genes in this plant.

Local conformational changes in the 8-17 deoxyribozyme core induced by activating and inactivating divalent metal ions.

The 8-17 deoxyribozyme (DNAzyme) is a catalytic DNA molecule capable of cleaving specific RNA substrates. The deoxyribozyme is activated by a wide variety of divalent metal ions, from Mg2+ to Pb2+, with just a few exceptions. It is not clear if metal ions are directly involved in catalysis, or are required to attain an active conformation, or both. In particular, the connection between metal-induced global structural rearrangements and catalysis is not straightforward. To gain more information on the local structural changes induced by metal ions, we introduced fluorescent 2-aminopurine (2-Ap) residues at different positions of the 8-17 'core'. We found that a construct containing 2-Ap at position 15 was best suited to monitor conformational changes in the presence of Mg2+, Ca2+ or Mn2+. Binding of these activating metal ions caused a local rearrangement at position 15, apparently entailing decreased stacking of the 2-Ap base. The metal dependence for such conformational change was generally hyperbolic (suggesting it mirrored the binding by a single metal ion) and yielded apparent dissociation constants close to those required for activation. In contrast, Cu2+, a divalent metal ion which does not support catalysis, caused in the deoxyribozyme a slow, reversible inactivation, which correlated with a very distinct conformational change at position 15.

Diversification of ß-Augmentation Interactions between CDI Toxin/Immunity Proteins.

Contact-dependent growth inhibition (CDI) is a widespread mechanism of inter-bacterial competition mediated by the CdiB/CdiA family of two-partner secretion proteins. CdiA effectors carry diverse C-terminal toxin domains (CdiA-CT), which are delivered into neighboring target cells to inhibit growth. CDI(+) bacteria also produce CdiI immunity proteins that bind specifically to cognate CdiA-CT toxins and protect the cell from auto-inhibition. Here, we compare the structures of homologous CdiA-CT/CdiI complexes from Escherichia coli EC869 and Yersinia pseudotuberculosis YPIII to explore the evolution of CDI toxin/immunity protein interactions. Both complexes share an unusual ß-augmentation interaction, in which the toxin domain extends a ß-hairpin into the immunity protein to complete a six-stranded anti-parallel sheet. However, the specific contacts differ substantially between the two complexes. The EC869 ß-hairpin interacts mainly through direct H-bond and ion-pair interactions, whereas the YPIII ß-hairpin pocket contains more hydrophobic contacts and a network of bridging water molecules. In accord with these differences, we find that each CdiI protein only protects target bacteria from its cognate CdiA-CT toxin. The compact ß-hairpin binding pocket within the immunity protein represents a tractable system for the rationale design of small molecules to block CdiA-CT/CdiI complex formation. We synthesized a macrocyclic peptide mimic of the ß-hairpin from EC869 toxin and solved its structure in complex with cognate immunity protein. These latter studies suggest that small molecules could potentially be used to disrupt CDI toxin/immunity complexes.

Reassimilation of ammonium in Lotus japonicus.

This review summarizes the most recent results obtained in the analysis of two important metabolic pathways involved in the release of internal sources of ammonium in the model legume Lotus japonicus: photorespiratory metabolism and asparagine breakdown mediated by aparaginase (NSE). The use of photorespiratory mutants deficient in plastidic glutamine synthetase (GS2) enabled us to investigate the transcriptomics and metabolomic changes associated with photorespiratory ammonium accumulation in this plant. The results obtained indicate the existence of a coordinate regulation of genes involved in photorespiratory metabolism. Other types of evidence illustrate the multiple interconnections existing among the photorespiratory pathway and other processes such as intermediate metabolism, nodule function, and secondary metabolism in this plant, all of which are substantially affected in GS2-deficient mutants because of the impairment of the photorespiratory cycle. Finally, the importance of asparagine metabolism in L. japonicus is highlighted because of the fact that asparagine constitutes the vast majority of the reduced nitrogen translocated between different organs of this plant. The different types of NSE enzymes and genes which are present in L. japonicus are described. There is a particular focus on the most abundant K(+)-dependent LjNSE1 isoform and how TILLING mutants were used to demonstrate by reverse genetics the importance of this particular isoform in plant growth and seed production.

Heme uptake in bacterial pathogens.

Iron is an essential nutrient for the survival of organisms. Bacterial pathogens possess specialized pathways to acquire heme from their human hosts. In this review, we present recent structural and biochemical data that provide mechanistic insights into several bacterial heme uptake pathways, encompassing the sequestration of heme from human hemoproteins to secreted or membrane-associated bacterial proteins, the transport of heme across bacterial membranes, and the degradation of heme within the bacterial cytosol to liberate iron. The pathways for heme transport into the bacterial cytosol are divergent, harboring non-homologous protein sequences, novel structures, varying numbers of proteins, and different mechanisms. Congruously, the breakdown of heme within the bacterial cytosol by sequence-divergent proteins releases iron and distinct degradation products.

The K+-dependent asparaginase, NSE1, is crucial for plant growth and seed production in Lotus japonicus.

The physiological role of K(+)-dependent and K(+)-independent asparaginases in plants remains unclear, and the contribution from individual isoforms during development is poorly understood. We have used reverse genetics to assess the phenotypes produced by the deficiency of K(+)-dependent NSE1 asparaginase in the model legume Lotus japonicus. For this purpose, four different mutants were identified by TILLING and characterized, two of which affected the structure and function of the asparaginase molecule and caused asparagine accumulation. Plant growth and total seed weight of mature mutant seeds as well as the level of both legumin and convicilin seed storage proteins were affected in the mutants. The mutants isolated in the present work are the first of their type in legumes and have enabled us to demonstrate the importance of asparagine and K(+)-dependent NSE1 asparaginase for nitrogen remobilization and seed production in L. japonicus plants.

Glutamine synthetase in legumes: recent advances in enzyme structure and functional genomics.

Glutamine synthetase (GS) is the key enzyme involved in the assimilation of ammonia derived either from nitrate reduction, N(2) fixation, photorespiration or asparagine breakdown. A small gene family is encoding for different cytosolic (GS1) or plastidic (GS2) isoforms in legumes. We summarize here the recent advances carried out concerning the quaternary structure of GS, as well as the functional relationship existing between GS2 and processes such as nodulation, photorespiration and water stress, in this latter case by means of proline production. Functional genomic analysis using GS2-minus mutant reveals the key role of GS2 in the metabolic control of the plants and, more particularly, in carbon metabolism.

Structural analysis of K+ dependence in L-asparaginases from Lotus japonicus.

The molecular features responsible for the existence in plants of K+-dependent asparaginases have been investigated. For this purpose, two different cDNAs were isolated in Lotus japonicus, encoding for K+-dependent (LjNSE1) or K+-independent (LjNSE2) asparaginases. Recombinant proteins encoded by these cDNAs have been purified and characterized. Both types of asparaginases are composed by two different subunits, α (20 kDa) and ß (17 kDa), disposed as (αß)2 quaternary structure. Major differences were found in the catalytic efficiency of both enzymes, due to the fact that K+ is able to increase by tenfold the enzyme activity and lowers the K(m) for asparagine specifically in LjNSE1 but not in LjNSE2 isoform. Optimum LjNSE1 activity was found at 5-50 mM K+, with a K(m) for K+ of 0.25 mM. Na+ and Rb+ can, to some extent, substitute for K+ on the activating effect of LjNSE1 more efficiently than Cs+ and Li+ does. In addition, K+ is able to stabilize LjNSE1 against thermal inactivation. Protein homology modelling and molecular dynamics studies, complemented with site-directed mutagenesis, revealed the key importance of E248, D285 and E286 residues for the catalytic activity and K+ dependence of LjNSE1, as well as the crucial relevance of K+ for the proper orientation of asparagine substrate within the enzyme molecule. On the other hand, LjNSE2 but not LjNSE1 showed ß-aspartyl-hydrolase activity (K(m) = 0.54 mM for ß-Asp-His). These results are discussed in terms of the different physiological significance of these isoenzymes in plants.

A threonine synthase homolog from a mammalian genome.

The genomes of several vertebrates contain two genes encoding proteins highly similar to threonine synthase (TS), even though the biosynthesis of l-threonine (l-Thr) is not known to occur in these animals. We report a bioinformatic analysis of the two TS-like genes, the recombinant expression of one murine TS homolog (mTSH2) and its initial biochemical characterization. Recombinant mTSH2 contained bound pyridoxal-5'-phosphate (PLP), but did not synthesize l-Thr. The enzyme did, however, bind O-phospho-homoserine (PHS; the actual TS substrate) and degraded it to alpha-ketobutyrate, phosphate, and ammonia-a known side reaction of microbial TSs. mTSH2 also degraded O-phospho-threonine (PThr) to alpha-ketobutyrate, showing that it can act as a catabolic phospho-lyase on both gamma- and beta-phosphorylated substrates. These findings suggest an unusual evolutionary origin for mTSH2, whereby an original TS enzyme became 'recycled' into a phospho-lyase upon dismissal, in metazoa, of the l-Thr biosynthetic pathway.

Kinetic and thermodynamic characterization of the RNA-cleaving 8-17 deoxyribozyme.

The 8-17 deoxyribozyme is a small DNA catalyst of significant applicative interest. We have analyzed the kinetic features of a well behaved 8-17 construct and determined the influence of several reaction conditions on such features, providing a basis for further exploration of the deoxyribozyme mechanism. The 8-17 bound its substrate with a rate constant approximately 10-fold lower than those typical for the annealing of short complementary oligonucleotides. The observed free energy of substrate binding indicates that an energetic penalty near to +7 kcal/mol is attributable to the deoxyribozyme core. Substrate cleavage required divalent metal ion cofactors, and the dependence of activity on the concentration of Mg2+, Ca2+ or Mn2+ suggests the occurrence of a single, low-specificity binding site for activating ions. The efficiency of activation correlated with the Lewis acidity of the ion cofactor, compatible with a metal-assisted deprotonation of the reactive 2'-hydroxyl group. However, alternative roles of the metal ions cannot be excluded, because those ions that are stronger Lewis acids are also capable of forming stronger interactions with ligands such as the phosphate oxygens. The apparent enthalpy of activation for the 8-17 reaction was close to the values observed for hydroxide-catalyzed and hammerhead ribozyme-catalyzed RNA cleavage.